Fanconi Anemia (FA) is an autosomal recessive disease caused by deficiency of proteins in a cellular pathway which impacts chromosome stability, DNA cross-link response, embryonic development, cancer susceptibility and stem cell survival. At least eight different genes are known to exist and three of these have been isolated. However, the biochemical function(s) of the FANC proteins are currently not understood. In order to understand the pathway's function and develop improved therapy for this disease, we will clone additional FANC genes and develop knock- out mice of these already cloned. We have narrowed the location of the FA group D gene to <300 kb and together with Project 1 will complete its positional cloning. In addition, we will use microcell-mediated chromosome transfer to map additional FA genes and positionally clone them in the future. We have generated knock-out mice for FA group C and will use these animals to study the role of FANCC in DNA cross-link responses in vivo, the biology of hematopoietic stem cells in FA and as a model FANCC in DNA cross-link responses in vivo, the biology of hematopoietic stem cells in FA and as a model for preclinical gene therapy. We will also generate knock-out mice for FA groups A and D. These mutants and their cells will be studied jointly with Projects 1 and 3. Mice doubly mutant in different FANC genes and genes involved in mammalian cross-link repair will be generated to study their cancer proneness hematopoiesis and responses to DNA damaging agents.